EP2869828B1 - Vault immuntherapie - Google Patents

Vault immuntherapie Download PDF

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Publication number
EP2869828B1
EP2869828B1 EP13816123.7A EP13816123A EP2869828B1 EP 2869828 B1 EP2869828 B1 EP 2869828B1 EP 13816123 A EP13816123 A EP 13816123A EP 2869828 B1 EP2869828 B1 EP 2869828B1
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vault
ova
cells
pharmaceutical composition
mvp
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French (fr)
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EP2869828A4 (de
EP2869828A1 (de
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Leonard H. Rome
Valerie A. Kickhoefer
Sherven Sharma
Steven M. Dubinett
Isaac YANG
Linda M. Liau
Kathleen A. KELLY
Jian Yang
Upendra K. Kar
Cheryl CHAMPION
Janina JIANG
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University of California
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University of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/0203—NAD+ ADP-ribosyltransferase (2.4.2.30), i.e. tankyrase or poly(ADP-ribose) polymerase
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
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    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
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    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
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    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore

Definitions

  • the present disclosure relates generally to the use of vault compositions as adjuvants for stimulating a cellular immune response to one or more antigens, for example, tumor antigens or cancer biomarkers. Also disclosed is the use of the compositions for the treatment of diseases, such as cancer.
  • the immune-promoting activity of any given vaccination strategy is determined by the presence of the relevant antigenic components in the vaccine formulation, enhanced by the addition of suitable adjuvants capable of activating and promoting an efficient immune response against infectious agents or cancers [1,2].
  • suitable adjuvants capable of activating and promoting an efficient immune response against infectious agents or cancers [1,2].
  • One approach for tailoring vaccines to elicit certain types of immune responses while avoiding inflammation is to develop subunit vaccines by combining non-living or synthetic antigens with adjuvants [9].
  • This type of vaccine can deliver defined antigens with reduced inflammatory cytokine production but is dependent on the adjuvant formulation to stimulate cell-mediated immune responses and protection from infectious challenge or prevent tumor growth [11,12].
  • Most licensed vaccines promote immunity by eliciting humoral immune responses and weak cellular immune responses. Current efforts are directed to producing adjuvants which elicit cell-mediated immunity [13,14].
  • MHC-I major histocompatibility complex
  • TLR toll-like receptor
  • Nanoparticle pharmaceutical carriers can be engineered to elicit various types of immunity and are increasingly investigated as adjuvants for vaccines.
  • Different types of nanocarriers such as polymers (polymeric nanoparticles, micelles, or dendrimers), lipids (liposomes), viruses (viral nanoparticles), and organometallic compounds (carbon nanotubes) have been employed for immunotherapeutic applications [21-23].
  • vaults can be produced to contain a bacterial antigen and induce adaptive immune responses and protective immunity following immunization [27].
  • vault nanocapsules can also be engineered to promote anti-tumor responses [28].
  • Vaults are cytoplasmic ubiquitous ribonucleoprotein particles first described in 1986 that are found in all eukaryotic cells ( Kedersha et al., J Cell Biol, 103(3):699-709 (1986 )).
  • Native vaults are 12.9 ⁇ 1 MDa ovoid spheres with overall dimensions of approximately 40 nm in width and 70 nm in length ( Kong et al., Structure, 7(4):371-379 (1999 ); Kedersha et al., J Cell Biol, 112(2):225-235 (1991 )), present in nearly all-eukaryotic organisms with between 10 4 and 10 7 particles per cell ( Suprenant, Biochemistry, 41(49):14447-14454 (2002 )).
  • vault function remains elusive although they have been linked to many cellular processes, including the innate immune response, multidrug resistance in cancer cells, multifaceted signaling pathways, and intracellular transport ( Berger et al., Cell Mol Life Sci, 66(1):43-61 (2009 )).
  • Vaults are highly stable structures in vitro, and a number of studies indicate that the particles are non-immunogenic ( Champion et al., PLoS One, 4(4):e5409 (2009 )). Vaults can be engineered and expressed using a baculovirus expression system and heterologous proteins can be encapsulated inside of these recombinant particles using a protein-targeting domain termed INT for vault INTeraction. Several heterologous proteins have been fused to the INT domain (e.g.
  • WO 2008/151197 A2 describes a fusion protein in which an NY-ESO-1 polypeptide is fused to the surface of a vault particle at both ends of the vault particle in order to achieve higher oligomeric structures compared to NY-ESO-1. It also describes the use of the NY-ESO-1 polypeptide-vault-fusion protein as a molecular adjuvant.
  • US 2009/304751 A1 describes the use of an immunogenic peptide of Chlamydia incorporated within a vault-like particle for immunizing a subject against Chlamydia infection.
  • the present invention relates to a pharmaceutical composition for use in treating cancer comprising a therapeutically effective amount of a vault complex and at least one pharmaceutically acceptable excipient, wherein a tumor antigen is encapsulated within the vault complex.
  • the present invention further relates to a method of preparing a vault complex for the pharmaceutical composition for use of the present invention comprising a) mixing an INT or INT fusion protein generated in insect Sf9 cells with a MVP or MVP fusion protein generated in insect Sf9 cells to generate a mixture; b) incubating the mixture for a sufficient period of time to allow formation of vault complexes, thereby generating the vault complex for the pharmaceutical composition for use of the present invention.
  • OVA ovalbumin
  • vault nanocapsules can be used as subunit vaccines which can generate both cellular and humoral immunity against antigens for human pathogens and cancer, which we have demonstrated for a number of tumor associated antigens.
  • the present disclosure provides a method for stimulating a cellular immune response in a subject, comprising administering to the subject an effective amount of an antigenic peptide or an antigenic fragment or variant thereof incorporated within a vault complex.
  • the present disclosure provides a pharmaceutical composition for preventing or treating a subject for cancer, comprising a tumor antigen or an antigenic fragment or variant thereof incorporated within a vault complex, and optionally at least one pharmaceutically acceptable excipient, sufficient to stimulate a cellular immune response.
  • the present disclosure provides a method of preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a tumor antigen or an antigenic fragment or variant thereof incorporated within a vault complex, sufficient to stimulate a cellular immune response.
  • the administering reduces tumor volume or tumor growth.
  • the antigenic peptide is a tumor antigen.
  • the vault complex comprises two or more vault complexes, in which each vault complex comprises two or more different antigenic peptides or antigenic fragments or variants.
  • one or multiple copies of the antigenic peptide can be fused to INT or MVP. If fused to MVP, the antigenic peptide can be fused to the N-terminus of MVP or to the C-terminus of MVP.
  • the INT comprises the amino acid sequence of SEQ ID NO: 2.
  • the vault complex comprises MVP, in which the number of MVP is 1-78. In some embodiments, the number of MVP is 78.
  • the vault complex further comprises VPARP or modified VPARP, or a portion of VPARP, or a modified portion of VPARP.
  • the cellular immune response is induction of CD8 + and CD4 + memory T-cells. In other embodiments, the cellular immune response is production of INF ⁇ .
  • Further embodiments comprise administering to the subject a vault complex containing a chemokine, in which the chemokine can be CCL21.
  • the administration can be with or without an antigen.
  • the present invention relates to a pharmaceutical composition for use in treating cancer comprising a therapeutically effective amount of a vault complex and at least one pharmaceutically acceptable excipient, wherein a tumor antigen is encapsulated within the vault complex.
  • the present invention further relates to a method of preparing a vault complex for the pharmaceutical composition for use of the present invention comprising a) mixing an INT or INT fusion protein generated in insect Sf9 cells with a MVP or MVP fusion protein generated in insect Sf9 cells to generate a mixture; b) incubating the mixture for a sufficient period of time to allow formation of vault complexes, thereby generating the vault complex for the pharmaceutical composition for use of the present invention.
  • vault or “vault particle” refers to a large cytoplasmic ribonucleoprotein (RNP) particle found in eukaryotic cells.
  • the vault or vault particle is composed of MVP, VPARP, and/or TEP1 proteins and one or more untranslated vRNA molecules.
  • Vault complex refers to a vault or recombinant vault that encapsulates a small molecule or protein of interest.
  • a vault complex can include all the components of a vault or vault particle or just a subset.
  • a vault complex with just a subset of the components found in vaults or vault particles can also be termed a "vault-like particle".
  • vault-like particles include: 1) MVP without VPARP, TEP1 and vRNA; 2) MVP and either VPARP or a portion of VPARP, without TEP1 and vRNA; 3) MVP and TEP1 or a portion of TEP with or without the one or more than one vRNA, and without VPARP; 4) MVP without VPARP, TEP1 and vRNA, where the MVP is modified to attract a specific substance within the vault-like particle, or modified to attract the vault complex to a specific tissue, cell type or environmental medium, or modified both to attract a specific substance within the vault complex and to attract the vault particle to a specific tissue, cell type or environmental medium; and 5) MVP, and either VPARP or a portion of VPARP, or TEP1 or a portion of TEP1 with or without the one or more than one vRNA, or with both VPARP or a portion of VPARP, and TEP 1, with or without the one or more than one vRNA, where one or more than one of the MVP, VPARP or
  • Vault targeting domain or “vault interaction domain” is a domain that is responsible for interaction or binding of a heterologous fusion protein with a vault protein, or interaction of a VPARP with a vault protein, such as a MVP.
  • INT domain is a vault interaction domain from a vault poly ADP-ribose polymerase (VPARP) that is responsible for the interaction of VPARP with a major vault protein (MVP).
  • VPARP vault poly ADP-ribose polymerase
  • MVP major vault protein
  • INT domain refers to a major vault protein (MVP) interaction domain comprising amino acids 1563 - 1724 of VPARP.
  • MVP major vault protein.
  • cp-MVP cysteine-rich peptide major vault protein.
  • VPNRP refers to a vault poly ADP-ribose polymerase.
  • TEP-1 is a telomerase/vault associated protein 1.
  • vRNA is an untranslated RNA molecule found in vaults.
  • vector is a DNA or RNA molecule used as a vehicle to transfer foreign genetic material into a cell.
  • the four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes.
  • Vectors can include an origin of replication, a multi-cloning site, and a selectable marker.
  • a "cell” includes eukaryotic and prokaryotic cells.
  • organ As used herein, the terms “organism”, “tissue” and “cell” include naturally occurring organisms, tissues and cells, genetically modified organisms, tissues and cells, and pathological tissues and cells, such as tumor cell lines in vitro and tumors in vivo.
  • extracellular environment is the environment external to the cell.
  • in vivo refers to processes that occur in a living organism.
  • a "subject" referred to herein can be any animal, including a mammal (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), a domestic animal (e.g., cat, dog, ferret, etc.), an avian species, or a human.
  • a mammal e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.
  • a farm or commercial animal e.g., a cow, horse, goat, donkey, sheep, etc.
  • a domestic animal e.g., cat, dog, ferret, etc.
  • an avian species e.g., an avian species, or a human.
  • mammal as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
  • the term "sufficient amount” is an amount sufficient to produce a desired effect, e.g., an amount sufficient to stimulate a cellular immune response.
  • terapéuticaally effective amount is an amount that is effective to ameliorate a symptom of a disease, such as cancer.
  • a “prophylactically effective amount” refers to an amount that is effective for prophylaxis.
  • stimulating refers to activating, increasing, or triggering a molecular, cellular or enzymatic activity or response in a cell or organism, e.g. a cellular immune response.
  • inhibiting refers to deactivating, decreasing, or shutting down a molecular, cellular or enzymatic activity or response in a cell or organism.
  • administering includes any suitable route of administration, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, including direct injection into a solid organ, direct injection into a cell mass such as a tumor, inhalation, intraperitoneal injection, intravenous injection, topical application on a mucous membrane, or application to or dispersion within an environmental medium, and a combination of the preceding.
  • treating refers to the reduction or elimination of symptoms of a disease, e.g., cancer.
  • preventing refers to the reduction or elimination of the onset of symptoms of a disease, e.g., cancer.
  • regressing or “regression” refers to the reduction or reversal of symptoms of a disease after its onset, e.g., cancer remission.
  • modified and variations of the term, such as “modification,” means one or more than one change to the naturally occurring sequence of MVP, VPARP or TEP1 selected from the group consisting of addition of a polypeptide sequence to the C-terminal, addition of a polypeptide sequence to the N-terminal, deletion of between about 1 and 100 amino acid residues from the C-terminal, deletion of between about 1 and 100 amino acid residues from the N-terminal, substitution of one or more than one amino acid residue that does not change the function of the polypeptide, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, such as for example, an alanine to glycine substitution, and a combination of the preceding.
  • the term percent "identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970 ), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988 ), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990 ). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
  • compositions disclosed herein are compositions disclosed herein.
  • compositions and methods of using vault complexes includes compositions and methods of using vault complexes.
  • An embodiment disclosed herein has recombinant vaults having a MVP and an antigen, e.g., a tumor antigen.
  • the vault complex can be used as an adjuvant for stimulating a cellular immune response to the antigen.
  • compositions disclosed herein comprise a vault complex.
  • a vault complex is a recombinant particle that encapsulates a small molecule (drug, sensor, toxin, etc.), or a protein of interest, e.g., a peptide, or a protein, including an endogenous protein, a heterologous protein, a recombinant protein, or recombinant fusion protein.
  • Vault complexes disclosed herein can include a tumor antigen.
  • Vaults e.g., vault particles are ubiquitous, highly conserved ribonucleoprotein particles found in nearly all eukaryotic tissues and cells, including dendritic cells (DCs), endometrium, and lung, and in phylogeny as diverse as mammals, avians, amphibians, the slime mold Dictyostelium discoideum, and the protozoan Trypanosoma brucei ( Izquierdo et al., Am. J. Pathol., 148(3):877-87 (1996 )).
  • Vaults have a hollow, barrel-like structure with two protruding end caps, an invaginated waist, and regular small openings surround the vault cap.
  • Vaults have a mass of about 12.9 ⁇ 1 MDa ( Kedersha et al., J. Cell Biol., 112(2):225-35 (1991 )) and overall dimensions of about 42 x 42 x 75 nm ( Kong et al., Structure, 7(4):371-9 (1999 )).
  • the volume of the internal vault cavity is approximately 50 x10 3 nm 3 , which is large enough to enclose an entire ribosomal protein.
  • Vaults comprise three different proteins, designated MVP, VPARP and TEP 1, and comprise one or more different untranslated RNA molecules, designated vRNAs.
  • the number of vRNA can vary.
  • the rat Rattus norvegicus has only one form of vRNA per vault, while humans have three forms of vRNA per vault.
  • the most abundant protein, major vault protein (MVP) is a 95.8 kDa protein in Rattus norvegicus and a 99.3 kDa protein in humans which is present in 96 copies per vault and accounts for about 75 % of the total protein mass of the vault particle.
  • MVP major vault protein
  • the two other proteins are each present in between about 2 and 16 copies per vault.
  • VPARP VPARP
  • INT domain VPARP
  • INT fusion proteins VPARP, INT domain, and INT fusion proteins
  • a vault poly ADP-ribose polymerase includes a region of about 350 amino acids that shares 28% identity with the catalytic domain of poly ADP-ribosyl polymerase, PARP, a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage.
  • VPARP catalyzes an NAD-dependent poly ADP-ribosylation reaction, and purified vaults have poly ADP-ribosylation activity that targets MVP, as well as VPARP itself.
  • VPARP includes a INT domain (major vault protein (MVP) interaction domain). The INT domain is responsible for the interaction of VPARP with a major vault protein (MVP).
  • a vault complex disclosed herein can include a INT domain.
  • the INT domain also referred to as mINT domain for minimal INT domain, is responsible for interaction of a protein of interest with a vault protein such as a MVP.
  • the INT domain is expressed as a fusion protein with a protein of interest.
  • a protein of interest can be covalently or non-covalently attached.
  • the INT of the vault complexes disclosed herein are derived from VPARP sequences. Exemplary VPARP sequences and INT sequences can be found in Table 1.
  • the INT can have the entire naturally occurring sequence or portions of the sequence or fragments thereof.
  • the INT has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the VPARP and/or INT sequences disclosed in Table 1.
  • the INT is derived from a human VPARP, SEQ ID NO:3, GenBank accession number AAD47250, encoded by the cDNA, SEQ ID NO:5, GenBank accession number AF158255.
  • the vault targeting domain comprises or consists of the INT domain corresponding to residues 1473-1724 of human VPARP protein sequence (full human VPARP amino acid sequence is SEQ ID NO:3).
  • the vault targeting domain comprises or consists of the mINT domain comprising residues 1563-1724 (SEQ ID NO: 2) of the human VPARP protein sequence.
  • the vault targeting domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 or 3.
  • a major vault protein (MVP) interaction domain can be derived from TEP1 sequences.
  • Such interaction domains can be termed, for example INT2, to distinguish them from a VPARP interaction domain.
  • INT2 a major vault protein interaction domain
  • One of skill in the art understands that the INT can have the entire naturally occurring sequence of the vault interaction domain in TEP 1 or portions of the sequence or fragments thereof.
  • a vault complex disclosed herein can include an MVP.
  • Exemplary MVP sequences can be found in Table 1.
  • the MVP can have the entire naturally occurring sequence or portions of the sequence or fragments thereof.
  • the MVP has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the MVP sequences disclosed in Table 1.
  • the MVP is human MVP, SEQ ID NO:6, GenBank accession number CAA56256, encoded by the cDNA, SEQ ID NO:7, GenBank accession number X79882. In other embodiments, the MVP is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the MVP sequences described herein.
  • a vault complex comprising, consisting essentially of, or consisting of an MVP modified by adding a peptide to the N-terminal to create a one or more than one of heavy metal binding domains.
  • the heavy metal binding domains bind a heavy metal selected from the group consisting of cadmium, copper, gold and mercury.
  • the peptide added to the N-terminal is a cysteine-rich peptide (CP), such as for example, SEQ ID NO:8, the MVP is human MVP, SEQ ID NO:6, and the modification results in CP-MVP, SEQ ID NO:9, encoded by the cDNA, SEQ ID NO: 10.
  • Any of the vault complexes described herein can include MVPs or modified MVPs disclosed herein.
  • a vault complex disclosed herein can include a TEP1 protein.
  • TEP1 sequences can be found in Table 1.
  • the TEP1 can have the entire naturally occurring sequence or portions of the sequence or fragments thereof.
  • the TEP1 has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the TEP1 sequences disclosed in Table 1.
  • the TEP1 can be human TEP1, SEQ ID NO: 11, GenBank accession number AAC51107, encoded by the cDNA, SEQ ID NO:12, GenBank accession number U86136. Any of the vault complexes described herein can include TEP1 or modifications thereof.
  • a vault complex disclosed herein can include a vRNA.
  • vRNA sequences can be found in Table 1.
  • the vRNA can have the entire naturally occurring sequence or portions of the sequence or fragments thereof.
  • the vRNA has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the vRNA sequences disclosed in Table 1.
  • the vRNA can be a human vRNA, SEQ ID NO: 13, GenBank accession number AF045143, SEQ ID NO:14, GenBank accession number AF045144, or SEQ ID NO:15, GenBank accession number AF045145, or a combination of the preceding.
  • any of MVP, VPARP, TEP1 and vRNAs can be from any species suitable for the purposes disclosed in this disclosure, even though reference or examples are made to sequences from specific species. Further, as will be appreciated by one of ordinary skill in the art with reference to this disclosure, there are some intraspecies variations in the sequences of MVP, VPARP, TEP1 and vRNAs that are not relevant to the purposes of the present disclosure. Therefore, references to MVP, VPARP, TEP1 and vRNAs are intended to include such intraspecies variants.
  • Suitable expression vectors generally include DNA plasmids or viral vectors.
  • Expression vectors compatible with eukaryotic cells can be used to produce recombinant constructs for the expression of an iRNA as described herein.
  • Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of expression vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • Plasmids expressing a nucleic acid sequence can be transfected into target cells as a complex with cationic lipid carriers (e.g. , Oligofectamine) or non-cationic lipid-based carriers (e.g ., Transit-TKOTM).
  • cationic lipid carriers e.g. , Oligofectamine
  • non-cationic lipid-based carriers e.g ., Transit-TKOTM
  • Successful introduction of vectors into host cells can be monitored using various known methods.
  • transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g ., antibiotics and drugs), such as hygromycin B resistance.
  • Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g.
  • the constructs can include viral sequences for transfection, if desired.
  • the construct may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors.
  • Constructs for the recombinant expression of a nucleic acid encoding a fusion protein will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the fusion nucleic acid in target cells.
  • regulatory elements e.g., promoters, enhancers, etc.
  • Vectors useful for the delivery of a nucleic acid can include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the nucleic acid in the desired target cell or tissue.
  • the regulatory elements can be chosen to provide either constitutive or regulated/inducible expression. A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the transgene.
  • viral vectors that contain the recombinant gene can be used.
  • a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993 )). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA.
  • the nucleic acid sequences encoding a fusion protein are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient.
  • retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994 ), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994 ); Kiem et al., Blood 83:1467-1473 (1994 ); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993 ); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993 ).
  • Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Patent Nos. 6,143,520 ; 5,665,557 ; and 5,981,276 .
  • Adenoviruses are also contemplated for use in delivery of isolated nucleic acids encoding fusion proteins into a cell.
  • Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia or for use in adenovirus-based delivery systems such as delivery to the liver, the central nervous system, endothelial cells, and muscle.
  • Adenoviruses have the advantage of being capable of infecting non-dividing cells.
  • Kozarsky and Wilson Current Opinion in Genetics and Development 3:499-503 (1993 ) present a review of adenovirus-based gene therapy.
  • a suitable AV vector for expressing a nucleic acid molecule featured in the present disclosure a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010 .
  • Adeno-associated virus AAV vectors
  • Walsh et al. Proc. Soc. Exp. Biol. Med. 204:289-300 (1993 ); U.S. Pat. No. 5,436,146 .
  • Suitable AAV vectors for expressing the dsRNA featured in the present disclosure, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101 ; Fisher K J et al. (1996), J. Virol, 70 :
  • a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
  • the pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • Examples of additional expression vectors that can be used in the present disclosure include pFASTBAC expression vectors and E. coli pET28a expression vectors.
  • recombinant vectors capable of expressing genes for recombinant fusion proteins are delivered into and persist in target cells.
  • the vectors or plasmids can be transfected into target cells by a transfection agent, such as Lipofectamine.
  • Examples of cells useful for expressing the nucleic acids encoding the fusion proteins of the invention include Sf9 cells or insect larvae cells.
  • Recombinant vaults based on expression of the MVP protein alone can be produced in insect cells. Stephen, A.G. et al. (2001). J. Biol. Chem. 276:23217:23220 ; Poderycki, M.J., et al. (2006). Biochemistry (Mosc). 45: 12184-12193 .
  • the present disclosure provides methods using pharmaceutical compositions comprising the vault complexes disclosed herein.
  • These compositions can comprise, in addition to one or more of the vault complexes, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • the pharmaceutical compositions that are injected intra-tumorally comprise an isotonic or other suitable carrier fluid or solution.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.
  • compositions for oral administration can be in tablet, capsule, powder or liquid form.
  • a tablet can include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
  • administration of the pharmaceutical compositions may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Formulations may be reconstituted from freeze-dried (lyophilized) preparations. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
  • Vault complexes described herein can be used to deliver a protein of interest (e.g., a tumor antigen) to a cell, a tissue, an environment outside a cell, a tumor, an organism or a subject.
  • the vault complex comprises a tumor antigen
  • the vault complex is introduced to the cell, tissue, or tumor.
  • the vault complex is introduced into the extracellular environment surrounding the cell.
  • the vault complex is introduced into an organism or subject. Delivery of the vault complex disclosed herein can include administering the vault complex to a specific tissue, specific cells, an environmental medium, or to the organism.
  • the methods disclosed herein comprise delivering a biomolecule to a cell by contacting the cell with any of the vault complexes described herein.
  • Cells disclosed herein can include, but are not limited to, any eukaryotic cell, mammalian cell, or human cells, including tumor cells.
  • Methods disclosed herein include delivery of the vault complex to a subject.
  • the delivery of a vault complex to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a vault complex to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the vault complex or components of the vault complex.
  • the vault complex is administered to a mammal, such as a mouse or rat. In another embodiment, the vault complex is administered to a human.
  • the methods of delivery disclosed herein include systemic injection of vaults. In other embodiments, the methods of delivery disclosed herein include oral ingestion of vaults.
  • the present disclosure features a method of treating or managing disease, such as cancer, by administering the vault complex disclosed herein to a subject (e.g., patient).
  • the method disclosed herein comprises treating of cancer in a subject in need of such treatment or management, comprising administering to the subject a therapeutically effective amount of the vault complexes described herein.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range of the vault complex. Such information can be used to more accurately determine useful doses in humans.
  • compositions according to the present disclosure to be given to a subject administration is preferably in a "therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980 .
  • a composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the dosage of vault complexes is between about 0.1 and 10,000 micrograms per kilogram of body weight or environmental medium. In another embodiment, the dosage of vault complexes is between about 1 and 1,000 micrograms per kilogram of body weight or environmental medium. In another embodiment, the dosage of vault complexes is between about 10 and 1,000 micrograms per kilogram of body weight or environmental medium.
  • the dosage is preferably administered in a final volume of between about 0.1 and 10 ml.
  • the dosage is preferably administered in a final volume of between about 0.01 and 1 ml.
  • the dose can be repeated a one or multiple times as needed using the same parameters to effect the purposes disclosed in this disclosure.
  • the pharmaceutical composition may be administered once to a subject, or the vault complex may be administered as two, three, or more sub-doses or injections at appropriate intervals.
  • the vault complexes can be injected in sub-doses in order to achieve the total required dosage.
  • the vault complexes featured in the present disclosure can be administered in combinations of vault complexes containing different tumor antigens, or in combination with other known agents effective in treatment of cancer.
  • An administering physician can adjust the amount and timing of vault complex administration or injection on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • the methods disclosed herein include preparing the vault complexes described herein.
  • the vault complexes are derived or purified from natural sources, such as mammalian liver or spleen tissue, using methods known to those with skill in the art, such as for example tissue homogenization, differential centrifugation, discontinuous sucrose gradient fractionation and cesium chloride gradient fractionation.
  • the vault complexes are made using recombinant technology.
  • a target of interest i.e., protein of interest
  • the target of interest may be selected from the group consisting of an enzyme, a pharmaceutical agent, a plasmid, a polynucleotide, a polypeptide, a sensor and a combination of the preceding.
  • the target of interest is a recombinant protein, e.g., a cell adhesion modifying substance, e.g., an RGD-containing peptide.
  • the polynucleotide sequences encoding the recombinant protein are used to generate a bacmid DNA, which is used to generate a baculovirus comprising the sequence.
  • the baculovirus is then used to infect insect cells for protein production using an in situ assembly system, such as the baculovirus protein expression system, according to standard techniques, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
  • the baculovirus protein expression system can be used to produce milligram quantities of vault complexes, and this system can be scaled up to allow production of gram quantities of vault complexes according to the present disclosure.
  • the target of interest is incorporated into the provided vaults.
  • incorporation is accomplished by incubating the vaults with the target of interest at an appropriate temperature and for an appropriate time, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
  • the vaults containing the protein of interest are then purified, such as, for example sucrose gradient fractionation, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
  • the vaults comprising the target of interest are administered to an organism, to a specific tissue, to specific cells, or to an environmental medium. Administration is accomplished using any suitable route, as will be appreciated by one of ordinary skill in the art with reference to this disclosure.
  • the method comprises preparing the composition disclosed herein by a) mixing a INT or INT fusion protein generated in insect Sf9 cells with a MVP or MVP fusion protein generated in insect Sf9 cells to generate a mixture; b) incubating the mixture for a sufficient period of time to allow formation of vault complexes, thereby generating the composition.
  • Sf9 cells are infected with pVI-MVP encoding recombinant baculoviruses. Lysates containing recombinant tumor antigen-INT and rat MVP generated in Sf-9 cells can be mixed to allow the formation of a macromolecular vault complex containing the tumor antigen-INT fusion protein.
  • Example 1 Preparation of recombinant vaults packaged with chicken ovalbumin
  • Recombinant vaults were produced using a baculovirus expression system in Sf9 insect cells that express a stabilized form of recombinant vaults (CP) and contain a cysteine rich peptide on the N terminus to increase stability [31]. Cryoelectron microscopy imaging of recombinant and tissue derived vaults revealed the localization of the MVP interacting domain, INT [31].
  • Another form of recombinant vaults (CPZ) contains a 33 amino acid mimic of the Ig binding domain of staphylococcal protein A (Z) in addition to the CP peptide [32].
  • CPZ vaults were shown to bind antibody and may direct uptake thorough FcRs [27].
  • FIG. 1 shows the co-purification of MVP and OVA-INT ( Figure 1 A) .
  • the identity of the components was confirmed by Western analysis with either an anti-MVP polyclonal antibody ( Figure 1B ) or an anti-OVA antibody ( Figure 1C ).
  • Purified CP-OVA recombinant vaults were evaluated by negative stain electron microscopy ( Figure 1D ).
  • Example 2 Ovalbumin packaged inside vault nanocapsules can induce a MHC-I restricted response
  • Dendritic cells possess the unique ability to process particulate antigens efficiently into the MHC-I pathway, in a process known as cross-priming.
  • Several approaches have been used to encourage cross priming such as adding exogenous antigenic proteins or peptides with adjuvants to stimulate cytotoxic T lymphocytes (CTLs) [35]. Therefore, we investigated whether recombinant vaults engineered to express OVA could be efficiently internalized, processed and presented by DC in an MHC-I restricted manner to activate CD8 + T cells.
  • the DC2.4 cell line (H-2K b ) was pulsed with CP-OVA and secretion of IL-2 was measured as an activation marker of the OVA-responsive CD8 + T cell hybridoma B3Z (H-2K b ).
  • the combination of DC2.4 cells, B3Z cells and CP that did not contain OVA-INT could not effectively stimulate IL-2 secretion.
  • CP-OVA produced by combining CP + OVA-INT incubated with both DC2.4 cells and B3Z hybridoma cells induced secretion of IL-2 ( Figure 2 ).
  • Example 3 Ovalbumin packaged inside vault nanocapsules can induce a MHC-II restricted response
  • OVA encased in vault nanoparticles at two concentrations 2.5 ⁇ g and 10.0 ⁇ g, stimulated a greater degree of T cell proliferation at both concentrations compared to recombinant OVA protein alone and were not statistically different from each other ( Figure 3 ).
  • Figure 3 show that OVA encased in vault nanocapsules was more effective at inducing CD4 + T cell proliferation than soluble OVA.
  • Example 4 Vaccination of mice with OVA packaged vault nanocapsules induces CD8 + and CD4 + T cells in vivo
  • the amount of OVA within the vaults and liposomes was quantitated by SDS gel quantitation ( Figure 4A ). Mice were immunized with equal amounts of delivery vehicle and OVA and the immunization regimen is described in Figure 4B .
  • the percentage of T cells responsive to the OVA CD8 peptide (SIINFEKL) or the OVA CD4 peptide 256-280 (TEWTSSNVMEERKIKV) were documented by surface, intracellular cytokine or perforin staining and FACS analysis after stimulation with each OVA peptide in C57BL/6 mice (H2 b background) as described in the methods section.
  • CD8+ T cells play a critical role in protection against viral and intracellular bacterial and protozoan infections and are important in tumor and graft rejection [39].
  • naive antigen (Ag)-responsive CD8 + T cells are able to proliferate quickly and differentiate into potent effector cells capable of rapid cytokine production and cytolytic killing of target cells [40,41].
  • OVA antigen-responsive CD8 + T cells
  • CD4 + T cell help is important for CD8 + T cell function. Since we observed increased numbers of OVA-responsive CD8 + memory and IFN ⁇ producing T cells in CP- and CPZ-OVA immunized mice, we investigated if the number of CD4 + T cells was also increased following vault immunization. To address this issue, splenocytes from each group were stimulated ex vivo with the class II peptide, OVA 265-280 and the CD4 + T cell response was characterized by FACS.
  • Example 5 Vault nanocapsules can be modified to induce select antibody Ig isotypes
  • mice immunized with Liposome-OVA induced significantly greater levels of anti-OVA IgG1 and IgG2c compared to CP-OVA, CPZ-OVA or OVA immunized mice ( Figures 7A & B ) indicating that liposomes induce high levels of anti-OVA antibody [44-46]. Further inspection revealed that the addition of the "Z" domain reduced mean anti-OVA IgG2c titers by 0.5 to 1 log in comparison to CP-OVA and OVA groups while IgG1 remained comparable.
  • mice immunized with vault nanocapsules modified to express the "Z" domain had a significantly increased this ratio compared to Liposome-OVA immunized group.
  • OVA and CP-OVA groups were not significantly different compared to the Liposome-OVA group ( Figure 7C ).
  • Example 6 Use of vault particles as an adjuvant to deliver an antigen
  • the vault particle When the vault particle is used as an adjuvant to deliver the model antigen ovalbumin (OVA) to mice harboring the solid tumor produced from Lewis lung carcinoma cells engineered to express ovalbumin, a cellular immune response directed against the tumor is induced resulting in immune attack on the tumor itself leading to reduction in the tumor size.
  • This antitumor immune response can be induced with a contralateral subcutaneous injection of the vault encapsulated ovalbumin with equal efficacy. See Figures 9A and B and 10A and B .
  • Example 7 Use of CCL21 chemokine containing vault particles to activate an antitumor response
  • the antitumor immune response to the vault adjuvant engineered to deliver specific antigens can be further activated by vault particles containing the CCL21 chemokine. See Figure 11A and B and Table 2.
  • the CCL21-vault can be combined with one or more than one vault containing tumor antigens to increase the cellular immune response induced toward the tumor. See Figure 11A and Table 2.
  • Example 8 Use of vault particles to deliver the tumor antigens
  • the vault particle When the vault particle is used as an adjuvant to deliver the tumor antigen NYESO1 to mice harboring the solid tumor produced from Lewis lung carcinoma cells engineered to express NYESO1, immune responses directed against the tumor are induced resulting in immune attack on the tumor itself. This antitumor immune response can be induced with a contralateral subcutaneous injection of the vault encapsulated NYESO. See Figure 12A and B .
  • GAA glioblastoma associated antigens
  • dendritic cell activation and maturation as measured by CD86 expression has also been shown to be significantly increased by treatment with NY-ESO vaults. See Figure 14 .
  • dendritic cells treated with GP100 vaults have demonstrated efficacy in stimulating CD8 T cells shown by elevated levels of interferon gamma. See Figure 15 .
  • Example 9 Use of vault particle delivery of tumor antigens for personalized therapeutics
  • compositions and methods disclosed herein can be utilized for personalized therapeutics directed against a wide variety of tumors. For example a biopsy of a particular tumor (lung glioblastoma etc.) can analyzed using existing procedures to determine the presence of common tumor antigens (biomarkers). Vault particles can be produced and engineered to contain individual tumor antigens and a mixture of these particles can be formulated based on the biopsy results of an individual tumor. This mixture of vault particles can then be used to immunize the patient and stimulate a specific cellular immune response that will be directed against the patient's particular tumor.
  • a biopsy of a particular tumor lung glioblastoma etc.
  • biomarkers common tumor antigens
  • Vault particles can be produced and engineered to contain individual tumor antigens and a mixture of these particles can be formulated based on the biopsy results of an individual tumor. This mixture of vault particles can then be used to immunize the patient and stimulate a specific cellular immune response that will be directed against the patient's particular tumor.
  • lung cancer there are approximately 10 to 15 different antigens (tumor biomarkers) that are primarily expressed in nearly 99% of all lung tumors.
  • Each of these 10 to 15 different antigens can be produced as fusion proteins with the vault packaging domain INT (antigen 1-INT, antigen 2-INT, antigen 3-INT etc.).
  • INT antigen 1-INT, antigen 2-INT, antigen 3-INT etc.
  • These 10 to 15 different antigens-INT fusion proteins can be expressed, purified and stored either separately or mixed with recombinant vaults to form individual vault adjuvant antigen preparations that can be stored.
  • an individual's lung tumor can be analyzed for expression of the presence of the common biomarkers (the 10 to 15 different antigens) that are present in that tumor, thus allowing for tailored treatments for tumor eradication.
  • a formulation of three different vault preparations (vaults containing antigen 3-INT, plus vaults containing antigen 5-INT plus vaults containing antigen 9-INT) can then be administered by subcutaneous injection to induce a cellular immune response to the individual tumor.
  • Recombinant baculoviruses were generated using the Bac-to-Bac protocol (Invitrogen, Carlsbad, CA).
  • the 385 amino acid coding region of ovalbumin was fused to major vault protein interaction domain (INT) derived from VPARP (amino acids 1563-1724) by PCR ligation[52,53].
  • INT major vault protein interaction domain
  • the second PCR reaction with primer OVA-INT forward TTGGCAGATGTGTTTCCCCTGCTAGCTGC ACACAACACTGGCAGGA and INT reverse: GGGCTCGAGTTAGCCTTGACTGTAATGGAG using INT in pET28 as the template.
  • the PCR reactions were purified on a Qiagen column and a second round of PCR was carried out using the OVA-forward x INT reverse.
  • the resultant PCR product containing the fused OVA-INT was purified on a Qiagen column, digested with Spe I and Xho I, gel purified, and ligated to pFastBac to form a pFastBac vector containing OVA-INT. Construction of cp-MVP-z, or cp-MVP in pFastBac has been described previously [32].
  • Sf9 cells were infected with Ova-INT, cp-MVP-z, or cp-MVP recombinant baculoviruses at a multiplicity of infection (MOI) of 0.01 for approximately 65 h and then pelleted and lysed on ice in buffer A [50 mM Tris-HCl (pH 7.4), 75 mM NaCl, and 0.5 mM MgCl2] with 1% Triton X-100, 1 mM dithiothreitol, 0.5 mM ' ⁇ g/ml chymostatin, 5 ⁇ M leupeptin, 5 ⁇ M pepstatin) (Sigma, St. Louis, MO).
  • MOI multiplicity of infection
  • Lysates containing cp-MVP-z vaults were mixed with lysates containing either OVA-INT were incubated on ice for 30 min to allow the INT fusion proteins to package inside of vaults.
  • Recombinant vaults were purified as previously described[33] and resuspended in 100-200 ⁇ l of sterile phosphate buffered saline.
  • the protein concentration was determined using the BCA assay (Pierce, Rockville, IL) and sample integrity was analyzed by negative stain electron microscopy and SDS-PAGE with Coomassie staining or transferred to hybond membrane (Amersham) for Western blot analysis.
  • the density of the bands was determined by gel scanning and densitometry analysis using a 9410 Typhoon Variable Mode Scanner (GE Healthcare Life Sciences, Piscataway, NJ).
  • DOTAP/DOPE (1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl- sn -glycero-3-phospho-ethanolamine) (Avanti Polar Lipids, Alabaster, AL) was re-hydrated in 1 mL endotoxin-free 5% glucose and mixed slowly (rotated) overnight at room temperature.
  • OVA-liposomes were separated from unincorporated ovalbumin by ultracentrifugation at 100,000 x g using an Optima XL-80K (Beckman Coulter, Fullerton, CA) ultracentrifuge and washed two additional times. Quantitation of encapsulated OVA was determined by subjecting OVA-liposomes (1, 2, 4 ⁇ L) to SDS-PAGE electrophoresis in parallel with known amounts of ovalbumin (0.25, 0.5, 1.0, 2.5, 5 ⁇ g) and visualized by Coomassie blue staining.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using the discontinuous buffer system and 4-15% acrylamide gels.
  • Protein samples of OVA-liposome or OVA-vaults were transferred to an Immobilon-P transfer membrane (Millipore, city, Bedford, MA) and blocked with 5% (wt/vol) nonfat dry milk in PBS-0.1% Tween 20 (PBS-T).
  • Membranes were incubated for 1 hr with anti-MVP (1:500, MAB 1023, Santa Cruz Biotechnology Inc, Santa Cruz, CA) or anti-INT followed by a 1 h incubation with the appropriate horseradish conjugate (1:5,000, Amersham Biosciences, Piscataway, NJ).
  • Bound conjugates were detected with ECL-Plus (GE Healthcare, Life Sciences, Piscataway, NJ) and 9410 Typhoon Variable Mode Scanner (GE Healthcare Life Sciences, Piscataway, NJ).
  • DC2.4 H-2Kb (5x10 4 /well) were plated in triplicates in 96-well plates and allowed to settle at 37 °C. Then, MHC Class I restricted CD8 + T cell line B3Z (10 5 /well) were added, in the presence of control vaults (200 ng/mL) and OVA vaults (200 ng/mL) for 24 hrs. After 24 h incubation at 37 °C, the plate was centrifuged at 1800 rpm, and the culture supernatant was collected and assayed for IL-2 using an IL-2 ELISA kit (BD Biosciences, San Jose, CA).
  • DC cultures were generated by flushing the bone marrow (BM) from the bone shafts, washed and plated bacteriological Petri dishes (Falcon Plastics, Oxnard, CA).
  • the cells were cultured at 2x10 5 cells/mL in RPMI 1640 culture medium (10 mM HEPES/2 mM 1-glutamine/10% 0.22 um filtered FBS/50 uM ⁇ -mercaptoethanol) supplemented with mGM-CSF (20 ng/mL) and mIL-4 (20 ng/mL) in an atmosphere of 5% CO 2 at 37 °C.
  • Fresh medium containing mGM-CSF (20 ng/mL) and mIL-4 (20 ng/mL) was added for 3-6 days after the start of culture.
  • Nonadherent cells consisting of mostly immature or mature DC were harvested for all the analyses performed in this study.
  • CD4 + T cells were separated from splenocytes with mouse CD4 + T-cell enrichment system (StemCell Technologies, Vancouver, Canada) according to the manufacturer's instructions.
  • CD4 + T cells (2 ⁇ 10 4 /well) were added to OVA protein or CP-OVA pulsed DC and cultured for an additional 4 days.
  • cells were pulsed with 1 ⁇ Ci [3H]thymidine (Amersham, Arlington, IL). The cells were harvested onto filter paper and [3H]thymidine incorporation was measured with a ⁇ -plate scintillation counter (PerkinElmer, Wellesley, MA).
  • the OVA protein concentration was adjusted using endotoxin-free sterile saline ( ⁇ 0.1 EU/mL, 1EU has ⁇ 0.1 of endotoxin (Sigma) to 2.5 ⁇ g OVA in 20 ⁇ g of vault nanoparticles or liposomes using a Typhoon 9410 Variable Mode Scanner of Coomassie blue stained SDS-PAGE gels.
  • the immunogens were injected into C57BL/6 mice (5-6 wk old) by subcutaneous injections at the base of the neck in 100 ⁇ l sterile saline. The mice were immunized 3 times at 2 wk intervals. The spleen and blood was obtained 2 wk after the last immunization. The splenocytes were immediately used for FACS analysis and serum samples were stored frozen at -80°C until assayed.
  • ELISA was used to determine the level of anti-OVA antibody isotypes in the serum. Briefly 96-well microtitre plates (Nunc, Roskilde, Denmark) were coated with 75 ⁇ l per well of OVA (1 ⁇ g /75 ⁇ l) in PBS and incubated over night at 4 °C. After being washed in buffer (phosphate buffered saline containing 0.05% Tween-20 (v/v) (PBS/T20) the plates were blocked with 150 ⁇ l of PBS supplemented with 5% non-fat dry milk for 2 h at room temperature. After washing, 7 ⁇ l of serum diluted from 1:40 to 1:5120 in PBS was added and incubated at 4 °C overnight.
  • buffer phosphate buffered saline containing 0.05% Tween-20 (v/v)
  • Unbound antibody was then washed away and 75 ⁇ l of goat antimouse IgG1- IgG2c-biotin (Southern Biotechnology Associates, Inc., Birmingham, AL), diluted 1/10,000 in PBS, was added and the plates incubated for 4 h at room temperature. The plates were then washed and 75 ⁇ l of NeutraAvidin horse radish peroxidase diluted in PBS at 1:1000 was added for 20 min. After a final wash step, 100 mL of TetraMethylBenzidine (TMB) (Zymed Laboratories Inc., San Francisco, CA) substrate was added and incubated at room temperature, in the dark, for 20 min. The reaction was stopped with 50 ⁇ L of 2 N sulphuric acid and the plates were read at 450 nm in a microplate reader (Model 550, Bio-Rad Laboratories, Hercules, CA).
  • TMB TetraMethylBenzidine
  • Spleens were removed and placed in RPMI media (Gibco, Grand Island, NY) supplemented with 10% heat inactivated FCS. They were macerated to release the lymphocytes which were then washed by centrifugation. The cell pellet was resuspended in fresh media at a concentration of 2 X 10 6 cells/mL and 1 mL of cells placed in each well of a 24-well plate (Nunc, Roskilde, Demark). They were restimulated with media (negative control) or OVA (100 ⁇ g/mL) for 72 h at 37°C in a humidified atmosphere with 5% CO 2 . The plate was frozen until required.
  • Lymphocytes were isolated from spleens by mechanical disruption through a cell strainer. RBCs were lysed using ammonium chloride-potassium buffer. The cells were stimulated @ 37°C with OVA peptide 265-280:TEWTSSNVMEERKIKV (2 ⁇ g) to identify CD4 cells or OVA peptide: SIINFEKL (2 ⁇ g) to identify CD8 cells for 5 hr. For the last 4h, cells were incubated in the presence of Brefeldin A (BioLegend) at 1 ⁇ g/mL.
  • the cells were stained using fluorochrome-conjugated MAbs against CD3, CD8, CD4, CD44, CCR7 and CD62L (BioLegend, San Diego, CA) in staining buffer (PBS with 2% fetal bovine serum and 0.1% sodium azide) and then treated with Fix/Perm (BioLegend). After permeabilization, the cells were further stained with fluorochrome-conjugated antibodies against IFN- ⁇ , IL-4, IL-17 and perforin. Data were collected on LSR II (BD Biosciences, San Jose, CA) and analyzed using FCS Express (De Novo Software, Los Angeles, CA).
  • CD8 + and CD4 + T cells were determined by gating on lymphocytes (FSC vs SSC) and CD8 + or CD4 + memory, cytokine producing or perforin expressing T cells were determined by gating on either CD3 + CD8 + or CD3 + CD4 + T cells as shown in Figure S1.
  • vault nanocapsules as adjuvants is the robust induction of CD8 + and CD4 + memory T cells.
  • vaults containing immunogenic proteins activate inflammasomes and escape into the cytoplasm [unpublished data, [27]. This may explain induction of an OVA-responsive CD8 + memory T cell response and cross-presentation. Vaults may also stimulate antigen-responsive CD8 + and CD4 + memory T cells by acting as intracellular depots or altering JAK/STAT signaling [47].
  • a potential vaccine should have the ability to induce and maintain antigen-responsive effector and/or memory T cells [7].
  • Our data show that immunization with vault nanocapsules was capable of inducing phenotypic markers of memory cells in CD8 + and CD4 + T cells. It will be interesting to extend these studies and examine memory responses in vivo using protection from infection or tumor models. In addition, we found enhanced production of OVA-responsive CD8 + T cells that could secrete IFN ⁇ . Surprisingly, there was not much difference between Liposome-OVA and OVA immunized groups and one questions the present of LPS. We did not measure LPS concentrations directly but all reagents used were endotoxin free and the purchased OVA was endotoxin free (see methods).
  • effector CD4 + T cells occurs in the same manner and with similar dynamics as is seen with the induction of effector memory CD8 + T cells [43].
  • the increased CD4 + memory T cells appear to be dominated by helper cells in mice immunized with CPZ-OVA vaults.
  • Our data shows that the addition of the "Z" domain modifies antibody isotypes and supports the increased ratio of anti-OVA IgG1 over IgG2c titers.
  • Adjuvants enhance immunity to immunogens but also steer immunity toward specific immune responses. For instance, alum is a known to promote Th2 responses [49].
  • the ability of vault vaccines to alter antibody isotypes suggests that modification of the vault toward certain immune responses is possible [50].
  • Vault nanocapsules act as "smart" adjuvants that are capable of directing immunity toward desired responses with little induction of inflammatory cytokines when delivered via a mucosal route [27]. Further studies comparing immunization routes will be needed to determine the most effective route for the desired immune response. Since vaults are ubiquitous and conserved across eukaryote species, the platform has a major advantage over other delivery systems which have safety concerns associated with attenuated bacteria or viruses.
  • vault nanocapsules are uniform in size and are able to be produced in abundance.
  • engineered vaults enhance the response with a much lower dose of the antigen and circumvent the protein-purification requirements of traditional subunit vaccines and particulate antigen-delivery modalities.
  • vaults provide a uniquely tunable platform with ease of manufacture for the delivery of a wide spectrum of subunit antigens for vaccines against infectious disease or other therapeutic targets.

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Claims (14)

  1. Pharmazeutische Zusammensetzung zur Verwendung bei der Behandlung von Krebs, die eine therapeutisch wirksame Menge eines Vault-Komplexes und mindestens einen pharmazeutisch verträglichen Hilfsstoff umfasst, wobei ein Tumorantigen innerhalb des Vault-Komplexes eingekapselt ist.
  2. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, wobei der Krebs ein Tumor ist und die Verabreichung der pharmazeutischen Zusammensetzung das Volumen des Tumors oder das Wachstum des Tumors reduziert.
  3. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1 oder Anspruch 2, wobei das Tumorantigen mit INT fusioniert ist.
  4. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 3, wobei die INT die Aminosäuresequenz der SEQ ID NO: 2 umfasst.
  5. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, wobei das Antigenpeptid mit einem Major Vault Protein (MVP) fusioniert ist.
  6. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, wobei der Vault-Komplex MVP umfasst.
  7. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 6, wobei die Anzahl der MVP 1-78, vorzugsweise 78, ist.
  8. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 6, wobei der Vault-Komplex weiterhin VPARP oder modifiziertes VPARP oder einen Teil von VPARP oder einen modifizierten Teil von VPARP umfasst.
  9. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, wobei die pharmazeutische Zusammensetzung eine zelluläre Immunantwort in einem Subjekt stimuliert, wenn sie dem Subjekt verabreicht wird.
  10. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 9, wobei die zelluläre Immunantwort eine Induktion von CD8+- und CD4+-Gedächtnis-T-Zellen ist.
  11. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 9, wobei die zelluläre Immunantwort eine Produktion von INFγ ist.
  12. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, die weiterhin einen Vault-Komplex umfasst, der ein Chemokin enthält, wobei das Chemokin vorzugsweise CCL21 ist.
  13. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 1, wobei die pharmazeutische Zusammensetzung zwei oder mehr Vault-Komplexe umfasst, wobei jeder Vault-Komplex zwei oder mehr verschiedene Antigenpeptide umfasst.
  14. Verfahren zur Herstellung eines Vault-Komplexes für die pharmazeutische Zusammensetzung zur Verwendung nach einem der Ansprüche 1-13, das a) Mischen eines in Sf9-Insektenzellen erzeugten INT oder INT-Fusionsproteins mit einem in Sf9-Insektenzellen erzeugten MVP oder MVP-Fusionsprotein, um eine Mischung zu erzeugen; b) Inkubieren der Mischung für eine ausreichende Zeitdauer, um die Bildung von Vault-Komplexen zu ermöglichen, wodurch der Vault-Komplex für die pharmazeutische Zusammensetzung zur Verwendung nach einem der Ansprüche 1-13 erzeugt wird, umfasst.
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